Abstract

Background and Purpose—Granulocyte colony–stimulating factor (GCSF) showed robust neuroprotective and neuroregenerative properties after stroke in rodents but failed to meet study end points in patients. Because immunologic side effects of GCSF may have escaped preclinical testing because of nonallometric dose translation, we hypothesized those as possible reasons.

Introduction

The peptide hormone granulocyte colony–stimulating factor (GCSF) was the most recent candidate for the treatment of stroke. Successfully tested in numerous rodent studies,1 it yielded neutral results in a large multicenter randomized clinical trial.2 Two issues may have jeopardized the translational process, both being potentially relevant for future research. First, preclinical doses cannot be linearly translated to patients. It was hence suggested to apply the body surface area normalization method to convert preclinical into clinical doses.3 Accordingly, back-calculating the GCSF dose that was used clinically (135 µg/kg)2 implies cumulative (3 days) GCSF dosages of 1665 µg/kg (mice) or 833 µg/kg (rats) for comparable and thus predictable rodent studies. These values were undermatched significantly in most preclinical trials with mean cumulative dosages of 50 µg/kg (mice) and 100 µg/kg (rats),1 illustrating a dose gap between preclinical and clinical application. Second, GCSF as a drug candidate for neurological disorders has been investigated predominantly from a CNS-centric viewpoint, but little is known on its immunologic effects during stroke pathophysiology.4 Because stroke outcome is significantly determined by sterile inflammation and systemic immune imbalance, GCSF as a key regulator of the immune system5 may unforeseeably influence these processes. We therefore decided to investigate whether and how GCSF dose adjustment influences poststroke inflammation in mice.

Materials and Methods

Experimental Stroke and Treatment Regime

Animal procedures were approved by local state authorities (TVV12/11) and performed according to the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines. A total of 72 adult male C57BL/6 mice were assigned randomly to 5 experimental groups: naive (n=3), sham (n=9), middle cerebral artery occlusion (MCAO) (n=20), GCSF low dose (n=20), and GCSF high dose (n=20). Animals were anesthetized with 2% isoflurane in a 30%O2/70%NO2 mixture, and focal cerebral ischemia was induced by filament occlusion (45 minutes) of the right middle cerebral artery. Mice received intraperitoneal injections of GCSF of either 50 or 832.5 µg/kg body weight distributed to 2 partial doses immediately after reperfusion and 12 hours later. GCSF treatment was controlled by application of 5% glucose in the others groups.

For details on further group allocation and end point measurements, see Methods in the online-only Data Supplement.

Results

Neurological Outcome and Peripheral Immunomodulation

The number of animals with high ranks (ie, more severe neurological deficit) in the categorical Bederson score was significantly increased by high-dose GCSF, whereas the infarct volume did not differ among MCAO groups (Figure 1A). When analyzing the impact of GCSF on circulating leukocyte populations, reduced numbers of T, B, and natural killer cells were found in all operated mice. Myeloid cells were not altered, but granulocytes (polymorphonuclear cells [PMN]) were significantly increased after GCSF treatment (Figure 1B). In the spleen, B cells decreased, whereas PMN increased because of surgery. T cells were significantly reduced after GCSF treatment. Again, myeloid cell counts were not influenced by surgery or treatment (Figure 1B). Next, we analyzed specific T-helper (Th) cell subpopulations revealing a decline of Th1 cells after MCAO that was reversed by high-dose GCSF. Th2 cells were decreased significantly because of surgery, but not further affected by MCAO or GCSF. Th17 cells showed a trend toward decrease after MCAO and increase after high-dose GCSF treatment. Regulatory T-cell counts were unaltered in the spleen (not shown) but significantly elevated in circulation after high-dose GCSF (Figure 1C). Serum levels of some cytokines were below (interferon-γ, tumor necrosis factor-α; not shown) or along (interleukin-1β; Figure 1D) the detection limit. Circulating interleukin-6 was significantly decreased by GCSF treatment irrespective of the dose. Interleukin-12/23p40 was not affected by GCSF. Determination of serum GCSF revealed a 10-fold difference between the dose groups (Figure 1D). We next differentiated the heterogeneous group of blood mononuclear myeloid cells by CD11c, major histocompatibility complex (MHC) II, and Ly6C expression. CD11c+/MHCII–/Ly6C– resident monocytes were significantly reduced after MCAO but restored by high-dose GCSF (Figure 2A, Q1). Circulating CD11b+/CD11c+/MHCII-/Ly6C+ dendritic cell (DC) precursors6 were doubled by high-dose GCSF (Q1). Blood myeloid (Figure 2A, Q2) and lymphoid (Figure 2B) DCs were not affected by MCAO or GCSF. Splenic CD8+ lymphoid DC were decreased by MCAO but not influenced by GCSF (Figure 2B). Ly6C+ inflammatory monocytes (Q4; MHCII–/CD11c–), including their activated variant (Q3, MHCII+/CD11c–), were also unchanged among experimental groups.

CNS Immunomodulation

MCAO caused a significant leukocyte influx into the ischemic hemisphere. Low-dose GCSF had no significant effect, but high-dose treatment tripled leukocyte counts within the brain (Figure 3A). This increase was not caused by PMN (Figure 3A) or B cells (not shown), but by T cells (Figure 3A) and mononuclear myeloid cells (Figure 3B). The latter population could be differentiated into MHCII+ cells, which primarily comprised macrophages, DC, and activated monocytes (Q2). Besides, MHCII–/Ly6C+ inflammatory monocytes were also increased in the GCSF high group (Q3). Finally, we found no significant effect of both GCSF doses on the gene expression of relevant chemoattractant factors and inflammatory cytokines in the ischemic hemisphere (Figure 3C).

Discussion

Our study indicates a considerable impact of high-dose GCSF treatment on poststroke immune responses, reflecting the GCSF dose given in a recent clinical trial2 when considering allometric dose retranslation.3 Immunomodulation induced by high-dose GCSF was further associated with worse neurological outcome. In contrast, GCSF dosing complying with most preclinical studies1 did not influence immune responses or functional outcome. We hypothesize that the surprising failure of GCSF in patients might, at least partly, be explained by dose-dependent immunologic side effects. This hypothesis is corroborated by the fact that patients with stroke receiving GCSF presented fever, tachycardia, monocytosis, and higher C-reactive protein levels.2 However, our findings are limited to the first 24 hours after stroke, and the relationship between dose-dependent immune alterations and neurological outcome requires confirmation in long-term studies.

Systemic effects of GCSF, such as increased mobilization of regulatory T cells, immature DC, and certain monocyte subpopulations, are in line with previous studies beyond the stroke field and may indicate a conducive dose-dependent gain of peripheral tolerance at larger time scales.5 During acute stroke, however, it was recently shown that regulatory T cells accumulate within the microvasculature and contribute to brain damage by compromising cerebral perfusion.7 Thus, the increase of circulating regulatory T cells and the compensation of stroke-induced Th1 cell depression8 may serve as possible explanations for detrimental effects of high-dose GCSF treatment early after stroke.

Another interesting finding was the distinct increase of monocytes/macrophages infiltrating into the ischemic lesion after high-dose GCSF treatment. We did neither observe shifts in splenic or circulating monocyte counts nor increased expression of chemoattractants that could explain this finding. One could speculate that high doses of GCSF induce functional changes of monocytes9 or modulate adhesion molecules10 on circulating cells and the CNS vasculature. It is also uncertain which consequences may arise from increased monocyte/macrophage counts in the ischemic lesion. A recent study in healthy mice indicated primarily beneficial effects,11 but the situation may turn in comorbid patients.12

In conclusion, we unveiled substantial immunomodulatory effects of GCSF at a dose corresponding to that used in a recent clinical trial. Within the first 24 hours after stroke, these changes seem to be detrimental, but long-term consequences are yet unknown. The delayed influence of high-dose GCSF on poststroke immune responses and functional outcome should be entirely understood before terminally amortizing GCSF as stroke treatment candidate. Application of lower doses to target neuroprotection while avoiding adverse immunomodulation or a timed administration (after the peak of thromboinflammation, but timely for regenerative effects) is a possible approach for future research.